SEQUENCE CLONING AND EXPRESSION ANALYSIS OF RAG GENES IN GOLD FISH

Recombination activating genes (Rag) are key genes in specific immunity system of vertebrate and also used asone of the molecularmarker for analysis of vertebrate evolution. Goldfish, Carassius auratus, with strong adaptive and diseases-resistant abilities, is one of the important freshwater economic fish extensively cultivated in China. And it also appears to be an excellentmodel animal for the evolution study of fish genome for its varied ploidy and abounding genetic diversity. The goldfish Rag genes have not been cloned and the specificity of goldfish specific immunity system has not been studied yet. In the present study, goldfish Rag 1 and Rag 2 geneswere cloned by polymerase chain reaction(PCR) methods, and the temporal and spatial expression pattern of RAG-1 were examined with methods of reverse transcription-polymerase chain reaction (RT-PCR) and whole mount in situ hybridization. The total length of the genomic sequence of the goldfish Rag 1gene from the initiation codon to the stop codon is4188 bp, which composed of three exons and two introns. The length of exon 1, 2 and 3 is 308bp, 1136bp and 1748bp respectively. The length of intron 1 and 2 is 105bp and 891bp respectively. The entire open reading frame (ORF) is 3192bp long, which predicated encoding a protein of 1063 amino acids. The goldfish Rag 2 has no intron in its open reading frame, the length of the genomic sequence from the initiation codon to the stop codon is 1593bp, which predicated encoding a protein of 530 amino acids. The putative structure of RAG protein in Carassius auratus are consisted of both an N2terminal domain and a core region. Comparative analysis onthe sequences of ORFS and protein amino acid of Carassius auratus, Danio rerio, Ctenopharyngodon idella, Cyprinus carpio and Oncorhynchus mykiss revealed that both Rag 1 and Rag 2 are highly conserved in evolution. Phylogenetic tree analysis on these fish based on Rag1 ORF suggested that Carassius auratus was more closely related to Cyprinus carpio, and which based on Rag 2 ORF indicated that Carassius auratus was more closely related to Danio rerio. The second intron of the Rag 1 was also highly conserved during evolution. Transcription factor binding sites analysis on the conserved region of this intron suggested that there were some putative transcription factor binding sites in it. A common conserved area which is appeared to be the SRY and SOX5 transcription factor binding siteswas observed in all the examined fish, suggesting that the expression of Rag 1 may have some relationship with the development of gonad. Tissue-specific expression analysis by RT-PCR methods, and it revealed that Rag 1 expressed mainly in the spermary and head kidney. This observation suggested that the RAGs directed DNA recombination took place not only in immunity tissue but also in gonads. Rag.expression was first detected at the 5d post-fertilization by RT-PCR. StrongmRNA in situ hybridization signalwas detected in the thymus primordium at the 9d post-fertilization, suggesting that this period might be an active DNA recombinationstage of immunogenes in goldfish.